Detection of light indicative of enzymatic activity from living organisms is a powerful tool in diagnostics, drug discovery and medicine that allows for the identification of disease pathways, determination of mechanisms of action, evaluation of efficacy of drug compounds, and monitoring lead candidates' effects on disease progression in living animals. For example, imaging myeloperoxidase activity has been described for monitoring oxidative stress. See, e.g., Gross et al. (2009) Nat Med 15:455-61; Kielland et al. (2009) Free Radic Biol Med. 47:760-6; and International Patent Publication WO 2010/062787). These methods use exogenously supplied luminol to detect invasive reactive oxygen species that are produced during inflammation processes by infiltrating neutrophils and monocytes/macrophages in stressed tissues.
Additional methods for imaging enzyme activity in vivo include bioluminescence resonance energy transfer (BRET) (see, e.g., Ward et al. (1978) Photochem. Photobiol. 27:389-396; Xu et al. (1999) Proc. Natl. Acad. Sci. USA 96:151-156; So et al. (2006) Nat. Biotechnol. 24:339-343) and Fluorescence by Unbound Excitation from Luminescence (FUEL) (see, e.g., Dragavon et al. 2010, Poster presentation at 10th International ELMI Meeting, Heidelberg) which use exogenous enzymes, such as luciferase, for light generation. Fluorescence resonance energy transfer (FRET), which investigates biological phenomena that produce changes in molecular proximity, uses exogenous excitation light.
Under physiological conditions, low levels of reactive oxygen and nitrogen species are generated by the cells and play a critical role in cell signaling and maintenance of vascular homeostasis. During pathological inflammatory responses, large quantity of reactive oxygen species (ROS) are generated by infiltrating neutrophils and monocytes/macrophages of stressed tissues. While there are a number of pathways for ROS production, one of them involves the membrane bound enzyme NADPH oxidase, which is abundant in neutrophils and monocytes/macrophages. Upon stimulation with inflammatory stimuli, the NADPH mediates a respiratory burst and converts O2 to superoxide anion (O°−), which is subsequently catalyzed by superoxide dismutase to form hydrogen peroxide (H2O2). Further reaction of hydrogen peroxide with the chloride anion is catalyzed by myeloperoxidase (MPO) to produce hypochlorous acid (HOCl). In the presence of luminol, oxidation of luminol by hypochlorous acid results in formation of 3-aminophthalate and accompanying light emission. Chemiluminescence light emission generated from this reaction cascade can be exploited as a read-out for superoxide production and myeloperoxidase activity.
However, detecting oxidative stress by imaging the luminol and hypochlorous acid reaction has limited sensitivity due to the short wavelength of the emitted light. Chemiluminescence light from the luminol oxidation reaction has a peak emission at 425 nm. It has been well established that tissue absorption of propagating light by chlorophores such as hemoglobin is most prominent with lower wavelength lights that are in the spectrum of 400-600 nm. As a result, luminol mediated chemiluminescence has limited penetration capability through tissues. Furthermore, there are concerns about immune reactions to the exogenous enzymes used in BRET and FUEL. FRET imaging methods are also problematic in that the exogenous excitation light can cause tissue autofluorescence which interferes with detection.
Therefore, there remains a need for compositions for and methods of monitoring enzymatic activity in living organisms.